SOLUBLE T-CELL ANTIGEN RECEPTORS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 466

localized immune cells were not stained with B-cell
marker-specific antibodies (i.e., anti-CD19 antibodies)
indicating that they are primarily NK cells. At the point
of peak NK cell infiltration, ALT-801 was also detected
with anti-huTCR antibody in the tumor sections, suggest-
ing that the fusion protein is associated with the immune
effector cells. In addition, the time course of NK cell
infiltration correlates well with the level of tumor growth
inhibition observed in TCR/IL-2 efficacy studies using this
xenograft tumor model [23].
Adoptive cell transfer experiments further verified the
tumor targeting effects of ALT-801 [23]. In vivo activated
splenocytes were isolated and incubated with ALT-801 or
MART1scTCR/IL-2 fusion proteins prior to transfer into
A375 tumor bearing nude mouse recipients. Significantly,
more accumulation of CD45R þ cells was observed in
tumors from nude mice receiving splenocytes incubated
with ALT-801 than in tumors from mice treated with sple-
nocytes preincubated with the control MART1scTCR/IL-2
(Figure 31.4C, D). In addition, when the tumor sections
were stained for either ALT-801 or MART1scTCR/IL-2
fusion proteins, significantly more cells bound with detect-
able levels of ALT-801 were observed infiltrating into the
tumors.
Based on these studies [23], a model has been proposed
in which ALT-801 first binds to the intermediate-affinity
IL-2 receptor, which is constantly expressed on NK and
other immune cells, and stimulates the cells to express
high- and intermediate-affinity IL-2 receptors. Subse-
quently administered ALT-801 binds these receptors to
form a stable complex and through the TCR domain
retargets the NK cells to the p53/HLA-A 0201 antigen
on the tumors. The activated NK cells then mediate their
cytotoxic effects against the tumors (Figure 31.4E).
The cytotoxic effects of the activated NK cells against
tumor may not need the assistance of the TCR once they
are inside the tumor. It has been established that NK cells
could be stimulated by stress proteins produced by cancer
cells through the NK cells activating receptors [44]. This
“cell-mediated” mechanism of action differs from that of
most therapeutic antibodies that rely on high-affinity inter-
action of soluble antibody with the tumor antigens. This
model also predicts that a high-affinity TCR may not be
necessary for efficacy if multiple copies of scTCRs bound
to the surface of activated NK cells provide sufficient
avidity to permit stable interactions with antigen-positive
target cells.

Fc dimerization fragment. We have demonstrated that the
p53-specific 264scTCR/IgG1 fusion was capable of binding
Fc receptors (FcR) on U937 human monocytic cells and p53
(aa 264-272)/HLA-A 0201 complexes on tumor cells [14].
This fusion protein was capable of promoting target cell and
effector cell conjugation and mediating effector cell killing
of target cells presenting p53 (aa 264-272)/HLA-A 0201
complexes by a mechanism similar to antibody-dependent
cellular cytotoxicity (ADCC).
An experimental metastasis lung model was used to
determine whether 264scTCR/IgG1 exhibits antitumor
activity in vivo [14]. Female athymic nude mice were
injected with the highly metastatic p53 þ /HLA-A 0201 þ
human melanoma subclone, A375-C15N, and tumors
were allowed to establish for 3 days. Mice were then treated
with varying doses of 264scTCR/IgG1, anti-STX mAb (an
isotype control mAb), or vehicle. Forty-two days after tumor
cell injection, lung nodules were counted. As shown in
Figure 31.5A, 264scTCR/IgG1 reduced lung metastasis
in a dose-dependent manner reaching maximum effect at
a dose of 1mg/kg, whereas the isotype control failed to
reduce metastasis. These data indicate that 264scTCR/IgG1
exhibits targeted activity in vitro and potent anti-metastatic
activity in vivo, confirming that replacement of the antibody
recognition domains with that of a scTCR can provide an
antibody-like molecule with novel tumor antigen specific
functionality [14].
Using a similar strategy to the development of ALT-801,
we have created a chimeric version of the 264scTCR/IgG1
fusion protein (ALT-802). Additionally, to assess the role
of IgG effector function in this context, we have generated
a variant of this protein (ALT-802LALA) that contains two
mutations in the Fc domain known to reduce FcR-binding
activity [45]. As expected, the ALT-802 fusion protein was
capable of binding FcR on U937 cells and directing human
immune effector cell cytolytic activity against p53 (aa 264-
272)/HLA-A 0201-positive target cells, whereas the ALT-
802LALA fusion exhibited decreased FcR binding and
little or no ADCC-like effector activity. The antitumor
activity of ALT-802 was examined in an NSCLC xenograft
model using a derivative of the p53 þ humanlungadeno-
carcinoma A549 cell line [46] that had been transfected to
express HLA-A2 0201 (A2-A549-L1). By 10-15 days
after i.v. injection into nude mice, the A2-A549-L1 cells
form lung nodules that closely resemble the clinical fea-
tures of human NSCLC. When mice were treated after lung
tumor establishment (10-day post-tumor cell injection)
with ALT-802 (3-10mg/kg), ALT-802LALA (3mg/kg),
or PBS (equivalent volume) i.v. twice-weekly for 3-4
weeks, ALT-802 administration reduced the number and
size of the pulmonary tumor nodules compared to PBS
treatment (Figure 31.5B) (J. Wen, unpublished data). ALT-
802LALA fusion protein did not exhibit this antitumor
effect consistent with its lack of ADCC-like activity. Mice

31.4.3 Preclinical Efficacy Studies of Anticancer
p53 Specific scTCRIgG1 Fusion Proteins

As indicated earlier, we have generated antibody-like fusion
molecules by linking the scTCR domain to IgG constant
domain, particularly the CH2-CH3 domains comprising the